JPH0587768A - Biosensor - Google Patents

Abstract

PURPOSE:To accurately measure the concn. of glucose in a liquid vital specimen. CONSTITUTION:When a sensor is brought into contact with a liquid vital solution, glucose being a substrate and ascorbic acid being an inhibitor transmits through a dense layer 2 and a porous layer 3 to diffuse to an immobilized enzyme membrane 4 to be oxidized by ascorbic acid oxidase 8 and glucose oxidase 9. At the same time, potassium ferricyanide being an oxidizing type dye 10 conjugated with glucose oxidase is reduced to potassium ferrocyanide and the concn. of glucose is known from the oxidizing current value required in the oxidation of reduced potassium ferrocyanide by a cathode 6.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrode type biosensor for measuring a membrane potential or a current value obtained by an electrode reaction.

[0002]

2. Description of the Related Art As an electrode type biosensor which can easily measure the glucose concentration in blood, Japanese Patent Application Laid-Open No. 60-221
The one disclosed in No. 350 is known. This biosensor has a structure in which a measuring electrode is provided with a glucose oxidase which oxidizes glucose and a porous body which carries an oxidative dye conjugated thereto.

The action is to bring a biological sample solution (blood) into contact with a porous body, oxidize glucose by glucose oxidase, simultaneously reduce an oxidized dye, and oxidize the reduced dye at an electrode. The glucose concentration is known from the required oxidation current value. And, since the oxidation current value includes the current value obtained by oxidizing the inhibitor such as ascorbic acid, the inhibitor is oxidized at the counter electrode, and the current value is subtracted from the oxidation current value at the measurement electrode. The true concentration of glucose is calculated.

[0004]

In the above-mentioned conventional biosensor, the current value required for oxidation of the inhibitor at the measurement electrode and the current value required for oxidation of the inhibitor at the counter electrode However, the inhibitor concentration at the measurement electrode and the inhibitor concentration at the counter electrode are not always the same, and an accurate concentration cannot be measured due to the difference in enzyme diffusion resistance.

[0005]

Means for Solving the Problems To solve the above problems, the present invention provides an electrode-type biosensor which includes a redox enzyme (glucose oxidase) that oxidizes a substrate such as glucose and an oxidative dye, and ascorbic acid. An immobilized enzyme membrane carrying an enzyme (ascorbate oxidase) that oxidizes the inhibitor was provided.

[0006]

[Function] By providing an immobilized enzyme membrane carrying an enzyme that oxidizes an inhibitor such as ascorbate oxidase,
Oxidation of the inhibitor at the electrode is eliminated.

[0007]

Embodiments of the present invention will be described below with reference to the accompanying drawings. Here, FIG. 1 is a cross-sectional view of a main part of the biosensor according to the present invention.

In the biosensor, the distal end of the cylindrical case 1 is a dense layer 2 in contact with a biological sample solution, and under the dense layer 2, an asymmetric porous layer 3, an immobilized enzyme membrane 4, and an oxygen permeable membrane 5 are successively arranged.
And a cathode electrode 6 made of platinum, and the case 1
The inside is filled with an electrolyte solution such as KOH, and further, an anode electrode 7 made of lead or the like is provided.

Here, the asymmetric porous layer 3 is made of cellulose acetate and has a thickness of about 60 μm. The dense layer 2 side is densely packed and the immobilized enzyme membrane 4 side is roughened so that molecules larger than glucose can enter. In addition to blocking, the glucose molecules once taken in are allowed to reach the immobilized enzyme membrane 4 quickly.

The immobilized enzyme membrane 4 is composed of a collagen membrane having a thickness of about 40 μm, and carries ascorbic acid oxidase 8 which oxidizes ascorbic acid, glucose oxidase 9 which oxidizes glucose and an oxidative dye 10 such as potassium ferricyanide. ..

In the illustrated example, glucose oxidase 9 was immobilized in a layer close to the cathode electrode 6 and ascorbate oxidase 8 was immobilized in a layer distant from the cathode electrode 6, but each enzyme was not divided into layers to form a membrane. They may be randomly arranged inside. As the enzyme that oxidizes the inhibitor, uricase may be immobilized in addition to ascorbate oxidase 8.

When the biological sample solution is brought into contact with the sensor as described above, glucose as a substrate and ascorbic acid as an inhibitor permeate through the dense layer 2 and the porous layer 3 and diffuse into the immobilized enzyme membrane 4. Oxidation is performed according to the reaction formula shown in (Chemical formula 1).

[0013]

[Chemical 1]

Simultaneously with the above, potassium ferricyanide, which is an oxidation type dye, is reduced to potassium ferrocyanide, and the glucose concentration is known from the oxidation current value required for oxidizing the reduced potassium ferrocyanide at the cathode electrode 6. However, in the present invention, ascorbic acid, which is an inhibitor, has already been oxidized by the time it reaches the electrode.Therefore, the above-mentioned oxidation current value is the current value required to oxidize the dye. The current value required for oxidation is not included. Therefore, the oxidation current value and the glucose concentration in the biological sample solution are in good agreement.

In the examples, the case where the glucose concentration is measured using the oxygen electrode has been described, but it is also possible to measure the glucose concentration using the hydrogen peroxide electrode. In this case, it is not necessary to immobilize ascorbate oxidase because the adverse effect of ascorbic acid can be prevented, but since uric acid mixed as an inhibitor produces hydrogen peroxide when oxidized, uric acid oxidase is used. It is necessary to immobilize a certain uricase.

[0016]

FIG. 2 is a graph comparing the present invention and the conventional example with respect to the relationship between the ascorbic acid concentration and the current value, and FIG. 3 shows the relationship between the present invention and the conventional example with respect to the relationship between the glucose concentration and the current value. It is the graph which compared. From FIG. 2, it can be seen that according to the present invention, the measured value is reliable until the concentration of ascorbic acid reaches about 3%, and from FIG. 3, the glucose concentration and the oxidation current value are well proportional to a high concentration. I understand.

As is apparent from the above description and graphs, according to the present invention, an immobilized enzyme membrane carrying an enzyme that oxidizes an inhibitor such as ascorbic acid or uric acid on both the measurement electrode and the counter electrode of an electrode type biosensor. Since such a substance is provided, these inhibitors are not oxidized on the electrode, and the oxidation current at the measuring electrode is in proportion to the concentration of the substrate as it is, so that the accurate concentration can be measured.

[Brief description of drawings]

FIG. 1 is a sectional view of an essential part of a biosensor according to the present invention.

FIG. 2 is a graph comparing the present invention with a conventional example regarding the relationship between the ascorbic acid concentration and the current value.

FIG. 3 is a graph comparing the present invention with a conventional example regarding the relationship between glucose concentration and current value.

[Explanation of symbols]

1 ... Case, 2 ... Dense layer, 3 ... Porous layer, 4 ... Immobilized enzyme membrane, 6 ... Cathode electrode, 8 ... Inhibitor oxidase, 9 ...
Substrate oxidase, 10 ... Oxidizing dye.

Claims (2)

[Claims] 1. A biosensor in which the substrate concentration in a sample is measured by detecting the oxygen concentration after oxidizing the substrate in the presence of oxidoreductase or the concentration of a reaction substance produced by the oxidation of the substrate. , This biosensor is an electrode-type biosensor that measures the membrane potential or the current value obtained by the electrode reaction, and it also oxidizes the redox enzyme that oxidizes the substrate and the oxidation dye that couples with the redox enzyme, as well as the inhibitor. A biosensor having an immobilized enzyme membrane carrying an enzyme. 2. The substrate is glucose, the redox enzyme is glucose oxidase, the inhibitor is ascorbic acid or uric acid, and the enzyme that oxidizes the inhibitor is ascorbate oxidase or uricase. The biosensor according to claim 1, wherein: